Action of Arthrobacter ureafaciens sialidase on sialoglycolipid substrates. Mode of action and highly specific recognition of the oligosaccharide moiety of ganglioside GM1.
Action of Arthrobacter ureafaciens sialidase on sialoglycolipid substrates. Mode of action and highly specific recognition of the oligosaccharide moiety of ganglioside GM1.
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脲酸节杆菌唾液酸酶对唾液酸糖脂底物的作用。
DOI：
10.1016/s0021-9258(18)36024-1
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
Y. Nagai
中科院分区：
文献类型：
--
作者：
M. Saito;K. Sugano;Y. Nagai
从产脲节杆菌（Arthrobacter ureafaciens）的培养滤液中分离出一种新的细菌唾液酸酶（N-乙酰神经氨酸糖水解酶，EC 3.2.1.18），详细描述了其对唾液酸糖脂的作用。强电解质对酶对脑神经节苷脂的作用具有可逆的抑制作用，这与离子环境对离子活性的Debye-Hückel效应一致，并导致酸性转变和pH最适范围的扩大。离子和非离子洗涤剂显着增强神经节苷脂上的酶活性，并导致这种酶的最适pH值的酸性转变。巯基似乎参与其活性部位。这种酶对唾液酸酶抗性神经节苷脂GM 1具有高度特异性作用，对GM 1的活性比产气荚膜梭菌唾液酸酶高约100倍，而产气荚膜梭菌唾液酸酶是迄今为止报道的唯一一种在胆盐存在下切割脂质底物的唾液酸酶。在不加去污剂的情况下，A.产脲菌唾液酸酶对GM 1的作用很低。单体或胶束形式的神经节苷脂GM 1被A.在约3倍于GM 1摩尔浓度的胆酸钠存在下，产脲杆菌唾液酸酶的活性最高。洗涤剂的存在下增加的Km和Vmax值神经节苷脂GM 1。在不加洗涤剂的条件下，这种唾液酸酶能很好地切割臭氧分解GM 1制备的寡糖，并且没有发现洗涤剂影响水解。糖底物的Km值比相应的脂质底物的Km值大约两个数量级。这表明，疏水神经酰胺部分增加了脂质底物对酶的亲和力，但抑制了底物的水解，这可能是由于其与酶分子的疏水部分的疏水相互作用（导致脂质底物的Km和Vmax较低）。由于唾液酸糖脂和去污剂分子的混合胶束的形成，这种抑制作用可被去污剂释放。研究还表明，单个唾液酸酶对GM 1的特异性糖结构的识别对于抗性唾液酸残基的释放是必需的，并且A.产脲菌唾液酸酶似乎具有同工酶或寡聚体结构。
A new bacterial sialidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) isolated from the culture filtrate of Arthrobacter ureafaciens was characterized in detail with respect to its action on sialoglycolipids. Strong electrolytes had a reversible inhibitory effect on the action of the enzyme on brain gangliosides in accordance with Debye-Hückel effect of ionic environment on ionic activity, and resulted in an acidic shift and a broadening of the pH optimum. Both ionic and non-ionic detergents markedly enhanced the enzymic activity on the gangliosides, and caused an acidic shift on the pH optimum of this enzyme. Sulfhydryl groups seemed to be involved in its active site. This enzyme had a highly specific action on sialidase-resistant ganglioside GM1, showing about 100-fold higher activity on GM1 than Clostridium perfringens sialidase, the only sialidase so far reported to cleave the lipid substrate in the presence of bile salts. In the absence of detergents, the activity of A. ureafaciens sialidase on GM1 was very low. Ganglioside GM1 in either the monomeric or micelar form was hydrolyzed to asialo-GM1 by A. ureafaciens sialidase most efficiently in the presence of sodium cholate of about three times the GM1 molar concentration. The presence of detergents increased both the Km and Vmax values for ganglioside GM1. The oligosaccharide prepared from GM1 by ozonolysis was cleaved well by this sialidase in the absence of detergents, and no detergent was found to affect the hydrolysis. The Km value for the sugar substrate was about two orders of magnitude greater than that for the corresponding lipid substrate. It is suggested that the hydrophobic ceramide moiety increases affinity of the lipid substrate to the enzyme, but inhibits hydrolysis of the substrate, possibly due to its hydrophobic interaction with hydrophobic portions of the enzyme molecule (resulting in lower Km and Vmax for lipid substrates). This inhibition may be released by detergent due to formation of mixed micelles of sialoglycolipid and detergent molecules. It is also indicated that recognition of the specific saccharide structure of GM1 by individual sialidases is essential for release of the resistant sialyl residue, and that A. ureafaciens sialidase seemed to have an isoenzymic or oligomeric structure.